ES2811255T3 - Bioprocess for the co-production of ethanol and mycoproteins - Google Patents

Abstract

An integrated method for producing and isolating mycoproteins produced by Fusarium venenatum and ethanol from a hydrolyzed starch raw material, said method comprising the steps of: a) providing an aqueous fermentable broth comprising a hydrolyzed starch raw material obtained from one or more cereals; b) fermenting at least a portion of the aqueous fermentable broth with Fusarium venenatum to obtain mycoproteins produced by Fusarium venenatum and a partially fermented broth; c) separating the mycoproteins produced by Fusarium venenatum from the partially fermented broth; d) fermenting at least a portion of the partially fermented broth, optionally with a portion of the non-fermented aqueous fermentable broth, with one or more microorganisms to obtain ethanol and a spent fermentation residue; and e) isolating the ethanol from the spent fermentation residue.

Description

DESCRIPTION

Bioprocess for the co-production of ethanol and mycoproteins

Field of the invention

The present invention relates to the co-production and isolation of mycoproteins and ethanol from hydrolyzed starch raw material (cereals).

Background of the invention

Cattle farms meet the growing consumer demand for meat and dairy products and use approximately 70% of the total global agricultural land for pasture and feed crop production. However, the growing population and changes in dietary preferences for more meat and dairy products will increase the demand for global agricultural land.

Biotechnology offers considerable potential to produce meat substitutes more efficiently with less environmental impact. A notable example is the mycoprotein (Quorn) produced by aerobic fermentation of glucose syrup. It is currently marketed as a healthy (relatively expensive) vegetarian alternative. Its relatively high cost is associated with the use of a refined raw material (glucose syrup) and the high fixed costs associated with the investment cost in an exclusively dedicated factory of modest capacity and the energy costs associated with aerobic fermentation.

Cattle feed is largely made up of cereals (corn, wheat) that provide the majority of carbohydrates, combined with protein enhancers (soy flour is the main source) to boost protein content for optimal nutrition . Approximately 40% of the grains are used as feed for cattle. The agricultural yield of soybeans is generally significantly lower than that of cereals.

Fermentation from cereal feedstock converts carbohydrates and leaves a protein-concentrated "distiller dried grains with solubles" (DDGS) residue. It is used as a high protein component in cattle feed, although its relatively low digestibility limits its mixing ratio.

Global bioethanol production is greater than 60 million tepa, with the 2 main sources being cereal fermentation (specifically, US corn converts approximately 40% of its production) and Brazilian cane sugar. The economic (excluding government incentives) and environmental benefits of bioethanol from cereals (corn / wheat) are marginal. There are significant political problems associated with food versus fuel pressures on land use and energy security.

US4447534 describes a process for producing ethanol using yeast, in which the control of growth conditions can increase the yield of yeast and hence yeast-derived single-cell proteins.

Silva et al. (Waste Management, 31 (2011), 108-114) describe a method to use the residue from the production of spirits and bioethanol for the production of proteins by yeasts, such as Saccharomyces cerevisiae and Candida parapsilosis. Single-cell protein from yeast can be used as a supplemental protein source for animal feed.

WO2009 / 079183 describes a process for improving the nutritional quality of a spent feed waste product that remains after grain fermentation to produce alcohol. The spent waste product can be fermented with a microbe that is capable of degrading cellulose and / or hemicellulose to produce one or more sugars and, in turn, using the sugar or sugars to proliferate. Since microbes contain proteins, their growth acts to increase the protein content of the spent waste product. A variety of microbes are described, including bacteria, yeast, and fungi, but no indication is given on a single cell protein isolated from the spent waste product.

Rasmussen et al., Bioresource Technology, 2014, vol. 151, pp. 284-290, describe a two-step method for ethanol production using corn as a carbohydrate feedstock and for producing mycoproteins with Rhizopus oligosporus.

Ferreira et al., Energies, 2014, vol. 7, pp. 3872-3885 present a study involving four food-grade fungi, for example, Fusarium venenatum, to investigate, among other questions, whether wheat-based fine distillery residues can be used to produce biomass and ethanol.

It would be desirable to provide a system that can be adapted to the production of mycoproteins and ethanol isolated from other material, while optionally varying the amount of each co-product based on desired requirements, such as prevailing economic and / or socio-economic problems.

Among the objects of the present invention is to provide a method for the production, optionally co-production, of isolated mycoproteins and ethanol.

Summary of the invention

In a first aspect, an integrated method is provided for producing and isolating mycoproteins produced by Fusarium venenatum and ethanol from a hydrolyzed starch raw material, said method comprising the steps of:

a) providing an aqueous fermentable broth comprising a hydrolyzed starch raw material obtained from one or more cereals;

b) fermenting at least a portion of the aqueous fermentable broth with Fusarium venenatum to obtain mycoproteins produced by Fusarium venenatum and a partially fermented broth;

c) separating the mycoproteins produced by Fusarium venenatum from the partially fermented broth;

d) fermenting at least a portion of the partially fermented broth, optionally with a portion of the non-fermented aqueous fermentable broth, with one or more microorganisms to obtain ethanol and a spent fermentation residue; Y

e) isolating the ethanol from the spent fermentation residue.

The carbohydrate raw material can be a mixed or a single raw material.

As mentioned above, the methods of the present invention are integrated and controllable in terms of the amount of mycoproteins or ethanol that can be produced. Through proper control of the substrate used to obtain the mycoproteins or ethanol and the growth conditions, especially the oxygen content, it is possible to vary, in a controllable way, the amount of mycoproteins or ethanol produced. Thus, for example, depending on prevailing commercial requirements, it is possible to vary the ratio of mycoproteins to ethanol that are produced according to the invention. This can be accomplished manually, semi-automatically, or fully automatic. For example, in an automatic or semi-automatic process, a user can indicate or enter into a user-programmable interface, for example, the desired ratio of mycoproteins to ethanol, and then the system can control the substrate flow and growth conditions. to achieve the required ratio of mycoproteins to ethanol.

Mycoproteins, as mentioned herein, are understood to be a form of unicellular protein specifically produced by Fusarium venenatum. The single raw material can comprise a plurality of metabolizable substrates, particularly cereal materials. The present method differs from non-integrated systems, which may require the supply of different raw materials for a mycoprotein production process and an ethanol production process.

Mycoproteins can be obtained through aerobic digestion of a substrate material. Ethanol can be produced through anaerobic fermentation of a substrate material. There may be an initial period of aerobic digestion to allow the growth of the microorganism or microorganisms that are capable of generating ethanol during anaerobic digestion.

In one embodiment, the microorganism or microorganisms for use in the production of mycoproteins and ethanol are different. In this embodiment, the microorganism or microorganisms capable of producing mycoproteins are a species of Fusarium, such as Fusarium venenatum, and the microorganism or microorganisms capable of producing ethanol are a species of Saccharomyces, such as Saccharomyces cerevisiae. The inventors have found that it is possible that a single type of microorganism can produce mycoproteins and then ethanol. Thus, in one embodiment, the integrated method of the invention employs a single type of microorganism to produce mycoproteins and ethanol. This only type of microorganism is F. venenatum.

The partially fermented broth can be derived from an initial fermentation broth that has undergone initial fermentation to produce mycoproteins. The partially fermented broth can be fermented to produce the second product, namely ethanol. In the present invention, mycoproteins are produced initially, followed by ethanol.

The methods of the present invention can be performed in a batch, continuous, or semi-continuous manner. The method may further comprise the step of separating the spent fermentation residue (also called distillery residue) to obtain a wet solids fraction and a soluble fraction. The soluble fraction can be concentrated, and the resulting syrup can be combined with the wet solids fraction, which can be dried to obtain a material known as dry distillers grains with soluble (DDGS).

Generally, said one or more cereal materials can include wheat, corn (maize), barley, rice, sorghum, buckwheat, oats, rye, and the like. The cereal can be food grade or it can be, in fact, a material which is no longer suitable for human consumption. One of the examples mentioned above can be used in the present invention, or mixtures comprising two or more types of cereals can be used. The present invention is not intended to be limited by the type or types of cereals that can be used, and these may simply be dictated by prevailing economic and / or geographical considerations and, therefore, availability at the time.

Said one or more cereal materials can be subjected to a process of milling, crushing and / or cutting to break the cereal material into smaller pieces and also to potentially release some of the proteins, sugars and other materials that may be present in the cereal. The degraded material can be mixed with water at a concentration, for example, 170-500 to 50 g / l, and the pH adjusted as necessary to provide the fermentation broth.

Any starch that may be present in the fermentation broth is subjected to hydrolyzing or partial hydrolyzing employing one or more of a gelatinization, liquefaction and / or saccharification. Starch occurs in nature as insoluble, non-dispersible granules resistant to enzymatic degradation. Gelatinization is the swelling of the starch granule in the presence of heat and water. At this point, the starch or shredded cereal slurry thickens considerably and would be difficult to process if alpha-amylases were not added to partially hydrolyze the starch to produce dextrins. The dextrin-containing solution is generally much more fluid or liquefied. Alpha-amylase acts to reduce the viscosity of the solution and also to produce a substrate with a smaller molecular size. A smaller molecular size substrate molecule is desired for the effective action of glucoamylase, which hydrolyzes dextrins to produce glucose.

Enzymes, such as alpha-amylase and glucoamylase, can be added to degrade or hydrolyze the starch that is present. Alpha-amylase is a bacterial thermostable endoamylase. Hydrolyzes α-1,4 bonds at random points on the starch molecule to rapidly reduce the viscosity of gelatinized starch solutions. This enzyme is a protein that contains metal ions and requires a small amount of calcium ions during use for maximum activity and stability. The enzyme cannot hydrolyze α-1,6 bonds, but it can circumvent these branch points in amylopectin. The product of the reaction are dextrins (short chains of glucose), and small amounts of glucose and maltose.

Glucoamylase, produced by fungi, is an exoamylase. Hydrolyzes maltose and dextrins from the non-reducing end of the molecule. Glucoamylase hydrolyzes both α-1,4 and α-1,6 linkages to completely degrade dextrins to glucose. The enzyme is optimally active at pH 3.5-4.5. Generally, alpha-amylase can be added at a concentration of 0.25-1.5% w / w of the solid material, and glucoamylase can be added at a concentration of 0.25-3% w / w of the solid material. . After enzymatic digestions, the fermentation broth can undergo a heat treatment to destroy the enzymes and kill any bacteria that may be present and interfere with the later stages of the process.

However, the addition of enzymes, pH adjustment, and necessary heating and cooling add many costs and therefore may be undesirable. The inventors have observed that microorganisms, such as Fusarium venenatum ( F. venenatum) can be fermented with a solution of non-hydrolyzed cereal grain starch. Since conventional mycoprotein production generally requires glucose as the starting carbon source, it was surprising that a material that had not undergone starch hydrolysis could be used to make mycoproteins. This can lead to considerable cost savings when the present process is carried out without the need for the liquefaction and / or saccharification steps.

The production of mycoproteins is carried out using F. venenatum to ferment a material within the fermentable broth. As mentioned above, the hydrolysis of the starch is carried out before carrying out the fermentation. An appropriate source of nitrogen and nutrients, such as Vogel salts, can be supplied for efficient mycoprotein fermentation (approximately 1 L of Vogel salts supplemented with 40 g of glucose). These can be added before and / or during fermentation.

It may be desirable to include an antifoam agent during fermentation to minimize any foaming that may occur due to, for example, proteins present in the fermentation broth. For example, rapeseed oil can be added at a concentration of up to 1% (by v / v). Not only can it serve as an antifoam agent, but rapeseed oil can also be used as a carbon source and can therefore be fermented. It may be necessary to add only an antifoam agent, such as rapeseed oil, at the beginning of the fermentation.

Being an aerobic fermentation process, the production of mycoproteins requires the addition of an oxygen source, in the form of air or oxygen. Along with controlling other fermentation conditions, the degree of aeration will influence the relative conversion of carbohydrates to mycoproteins and ethanol, and can be used to influence the conversion ratio. The integrated process that produces both products (mycoproteins and ethanol) allows operating conditions that do not require the minimization of ethanol (which would be a waste by-product in conventional mycoprotein fermentation) during mycoprotein fermentation. Conditions favoring mycoprotein / ethanol production can be selectively altered in the modes of sequential or phase operation. Desirably, operating conditions can be programmed in phases to correspond to aerobic fermentation to mycoproteins, which uses more energy, and the energy supply can change to correspond with the daily energy cycles of renewable energy availability.

The mycoprotein and / or ethanol fermentations can be carried out as batch, semi-continuous or continuous processes. A continuous process can offer advantages in the ability to maintain optimum constant state control conditions and interface with continuous separation processes. After completion, the spent fermentation broth and mycoproteins can undergo a heat treatment to remove / destroy nucleic acids, such as RNA, that may be present. The mycoproteins can then be separated / isolated from the spent fermentation broth, such as by centrifugation or filtration, for example, and then dried. The dried mycoprotein material can then be processed to provide a suitable food grade material. Spent mycoprotein fermentation liquors (combined with a different starch hydrolyzate and / or different fiber / protein solids) can be further fermented for ethanol production. S. cerevisiae can be used to ferment the material and produce ethanol. A typical process is described in Finn et al. (2006).

After having carried out the fermentation and having produced ethanol, it is necessary to separate / isolate the ethanol from the rest of the material that is present. This can be achieved by a continuous distillation process, as described, for example, in Cardona and Sánchez, 2007. This provides a high purity of ethanol, which can be further purified by passing it through molecular sieves, for example, to remove the water and concentrate the ethanol further.

The non-ethanol-containing material that is removed during the distillation process includes solids and a soluble material that is generally referred to as stills. These solids and soluble material can be separated by pressing or centrifugation, for example, to provide a wet solids material and a liquid. The wet solids material can then be dried, and the liquid can be concentrated to provide a syrup. The dried solids and syrup can be used separately or combined to provide a material known as dry distillers grains with soluble (DDGS).

It can be seen that the present invention provides a process that is capable of converting relatively low quality / value material into mycoproteins, which can be used as a source of human food; ethanol, which can be used as a biofuel; and spent material, such as DDGS, that can be used as animal feed.

Detailed description

The present invention will now be further described by way of non-limiting examples and referring to the figures showing:

Figure 1 shows a schematic flow diagram of a typical setup of an existing method for ethanol production; Figure 2 through Figure 8 show a schematic flow diagram of a series of exemplary method setups, by which mycoproteins and ethanol are co-produced from a grain feedstock.

Figure 1 - Flow diagram of a typical setup of an existing method for ethanol production.

Figure 2 - Flow diagram of an integrated setup of a co-production method for the production of mycoproteins and ethanol using two microorganisms.

Figure 3 - Another flow diagram of an integrated setup of a co-production method for the production of mycoproteins and ethanol using two microorganisms.

Figure 4 - Another flow diagram of an integrated setup of a co-production method for the production of mycoproteins and ethanol using two microorganisms and different raw materials.

Figure 5 - Flow diagram of a possible integrated setup of a co-production method for the production of mycoproteins and ethanol using a single microorganism (aerobic fermentation, followed by anaerobic fermentation).

Figure 6 - Flow diagram of an integrated setup of a co-production method for the production of mycoproteins and ethanol using a single microorganism (anaerobic fermentation, followed by aerobic fermentation). Figure 7 - Flow diagram of an integrated setup of a co-production method for the production of mycoproteins and ethanol using a single microorganism.

Figure 8 - Flow diagram of an integrated setup of a co-production method for the production of mycoproteins and ethanol using a single microorganism (anaerobic fermentation, followed by aerobic fermentation).

Figure 9 - Comparison of the growth of F. venenatum in minimal Vogel medium (flasks 1-3) and wheat hydrolyzate supplemented with Vogel salts (flasks 4-6). Shake flasks were incubated on an orbital shaker at 30 ° C and 150 rpm. The experiment was carried out in triplicate, as well as the calculation of the biomass. Samples were taken after 72 h (experiment 1).

Figure 10 - Batch fermentation of F. venenatum (Vw = 1.5 l) with wheat hydrolyzate supplemented with Vogel salts. The trend of dry biomass (•), glucose (▲) and ethanol (x) is shown throughout the fermentation (experiment 1).

Figure 11 - Growth of F. venenatum in 70g / l of hydrolyzate containing flour without enzymes (•), with 0.5% in w / w of α-amylase (A) and with 0.5% in w / w of α-amylase and 1% w / w glucoamylase (□) grown in shake flasks at 28 ° C and 150 rpm on an orbital shaker. Samples were taken after 24, 48 and 72 hours and analyzed in triplicate for biomass content (experiment 1).

Figure 12 - Optical density (OD) and dry cell weight of shake flasks of S. cerevisiae with MYPG and wheat hydrolyzate supplemented with Vogel salts. The flasks were incubated at 30 ° C and 250 rpm, and all contained 20 g / L glucose as a carbon source. They were cultured and analyzed in triplicate (experiment 2).

Figure 13 - Fermentation of ethanol with S. cerevisiae using MYPG and wheat hydrolyzate, respectively. Optical densities are shown throughout fermentation. MYPG was used in fermenter 1 (•) and fermenter 2 (o), while wheat hydrolyzate supplemented with Vogel salts was used in fermenter 3 (□) and fermenter 4 (A). Aeration was 0.82 vvm between 8-18 h, the rest 0 vvm. The temperature was kept constant at 30 ° C and the pH at 5.5 (experiment 2).

Figure 14 - Fermentation of ethanol with S. cerevisiae using MYPG and wheat hydrolyzate, respectively. The increase in dry biomass throughout fermentation is shown. MYPG was used in fermenter 1 (•) and fermenter 2 (o), while wheat hydrolyzate supplemented with Vogel salts was used in fermenter 3 (□) and fermenter 4 (A). Aeration was 0.82 vvm between 8-18 h, the rest 0 vvm. The temperature was kept constant at 30 ° C and the pH at 5.5 (experiment 2).

Figure 15 - Trend of dry cell weight (DCW) and glucose for the first fermentation of the integrated process with F. venenatum (experiment 3).

Figure 16 - Trends in dry cell weight (DCW), glucose and ethanol for the second part of the bioprocess integrated with S. cerevisiae.

Figure 17 - Comparison of dry cell weight trends of Fusarium venenatum fermentations with the application of different aeration profiles (experiment 4).

Figure 18 - Ethanol production throughout the fermentations of Fusarium venenatum with a variable oxygen supply (experiment 4).

Figure 19 - Trend of glucose concentrations throughout the fermentations of Fusarium venenatum with different fermentation media and different oxygen supply (experiment 4).

Figure 20 - Dissolved oxygen profile (DO,%) during the fermentations of F. venenatum (experiment 4).

The following key is used to interpret the various components and steps that are performed in the processes exemplified in Figures 1-8:

Grinding the grain physically breaks the grain to allow for starch extraction in downstream processing;

Liquefaction is a process to extract and hydrolyze starch from grain to obtain water soluble carbohydrates suitable for physical processing and fermentation. The grain is mixed and heated with water, usually with the use of enzymes. The heating process further sterilizes the solution for fermentation;

Separation of solid proteins and fiber by decantation, filtration, or centrifugation is performed to provide a soluble carbohydrate solution suitable for mycoprotein fermentation. This stage is not necessary in the conventional ethanol biorefining, since the ethanol, product of the fermentation, can be distilled from the suspension;

Aerobic fermentation provides for the fermentation growth of mycoproteins. This operation can be carried out in batch or continuous mode. A source of nitrogen (eg, ammonia), nutrients, and air / oxygen are introduced to the fermenter with cooling under controlled conditions to promote the growth of mycoproteins. Conditions can be controlled to optimize mycoprotein selectivity / growth rate or to achieve a preferred ratio of mycoproteins to ethanol by manipulating the physiological conditions through process variables, such as substrate flow or / and oxygen level; Heat treatment and isolation of the mycoprotein fermentation mixture provides a dried and isolated solid bulk mycoprotein product (by filtration or centrifugation). Heat treatment of the mixture is performed to reduce the nucleic acid (RNA) content of the product;

Anaerobic fermentation is done to convert residual carbohydrates into ethanol. This can be accomplished using either or both of mycoproteins and conventional brewer's yeast (eg, S. cerevisiae); Distillation is used to separate ethanol from fermentation mixtures;

Drying removes residual water from distilled ethanol to meet bioethanol fuel requirements. This is done, in a conventional way, using molecular sieves;

Isolation of the solid protein / fiber from the grain produces a high protein product that is generally used as feed for cattle. When isolated and dried together with the evaporated residue from distillation incorporating the soluble components of the fermentation mixtures, it is conventionally referred to as dry distiller's grains with soluble (DDGS).

As can be seen from the various processes exemplified in -8, the present invention provides a separation phase in which insoluble materials, such as protein and fiber, are removed to provide a solution comprising solubilized carbohydrates suitable for subsequent fermentation. .

In the various methods shown in Figures 2-8, an integrated process is provided that can produce ethanol and mycoproteins, as well as a spent waste material, dry distiller's grains with soluble (DDGS), that can be used as an animal feed product. .

Examples section

Experimental procedures

1.1 Microorganisms and culture conditions

The microorganism used in the mycoprotein (and ethanol) fermentations was the filamentous fungus F. venenatum A3 / 5 (20334), purchased from ATCC. Master cultures were produced by inoculating liquid cultures containing minimal Vogel's medium with 40 g / l glucose from potato dextrose agar (PDA) plates. They were incubated for 72 h at 150 rpm and 28 ° C on an orbital shaker before adding 20% glycerol to the cultures and storing them at -80 ° C in cryovials. In addition, the PDA plates were grown at 30 ° C and 0% CO ² in an incubator and then stored at 4 ° C.

For the ethanol fermentation the yeast S. cerevisiae (supplied from the Strathclyde Institute of Pharmacy and Biomedical Sciences culture collection) was also used. It was cultivated in MYPG medium with 1 L containing: 3 g of malt extract, 3 g of yeast extract, 5 g of peptone and 10 g of glucose, the glucose being added after autoclaving to avoid the Maillard reaction. The pH was adjusted to 5.5 and 15 g / l of agar was added to solidify the medium. The plates were inoculated with a loop and incubated for 24 h at 30 ° C in an incubator and then stored at 4 ° C. Master cultures were produced by inoculating a liquid culture and incubating it for 24 h on an orbital shaker at 30 ° C and 250 rpm before adding 20% glycerol and storing at -80 ° C in cryovials.

1.2 Preparation of the inoculum

The F. venenatum inoculum was prepared by inoculating a thawed vial from the cell bank at -80 ° C into a 200 ml aliquot of Vogel's medium. The culture was incubated at 30 ° C for a period of 24 hours. A 50 ml aliquot of this culture was removed (obtaining a 10% v / v inoculum) and transferred to a centrifuge tube. This was centrifuged at 8000 rpm for a period of 10 minutes. The supernatant was removed and an equal volume of sterile distilled water was added to resuspend the F. venenatum pellet. This was then centrifuged as before at 8000 rpm for a period of 10 minutes. The supernatant was again removed and the Fusarium venenatum pellet was resuspended in a 20 ml aliquot of sterile wheat medium. This aliquot was used to inoculate the bioreactor through the septum syringe port.

The preparation of the S. cerevisiae inoculum followed a similar strategy. A vial from the cell bank was thawed and used to inoculate a 200 ml flask containing MYPG medium. When the cell density reached sufficient levels (optical density of approximately 1 Au), a 50 ml aliquot of the culture was centrifuged, washed with distilled water and resuspended in wheat medium following the same procedure that was carried out with the inoculum of F. venenatum.

1.3 Preparation of the medium

1.3.1 Defined medium

The defined medium used for fermentation and shake flasks was prepared according to Vogel (1956). The sucrose carbon source was replaced by glucose and 40 g / l was used instead of 15 g / l.

1 liter of medium contained: 2.17 g of trisodium citrate, 5 g of potassium dibiphosphate, 2 g of ammonium nitrate, 0.2 g of magnesium sulfate heptahydrate, 0.1 g of calcium chloride dihydrate, 0 , 25 mg of biotin, and 5 ml of solution of trace elements.

For the dissolution of trace elements, the following trace elements were dissolved in 95 ml of distilled water: 5 g of citric acid monohydrate, 5 g of zinc sulfate heptahydrate, 1 g of ferrous ammonium sulfate hexahydrate, 0.25 g of cupric sulfate pentahydrate , 0.05 g of manganese sulfate monohydrate, 0.05 g of boric acid, and 0.05 g of sodium molybdate dihydrate.

1.3.2 Wheat hydrosylate (WH)

The wheat grain was ground to produce a flour material using an IKA analytical mill. This flour was dissolved in distilled water heated to 90 ° C with a final flour concentration in the region of 70 g / l. Already in solution, the pH of the system was adjusted to 7 before the addition of the enzyme α-amylase at a load of 1% w / w of the wheat flour added. The solution was kept at 90 ° C and stirred for a period of 1 hour. The solution was then allowed to cool while stirring until a temperature in the region of 50 ° C was reached. Then the pH of the solution was adjusted to 4.6-4.8 using 1M hydrochloric acid. Then the glucoamylase enzyme was added again with a loading of 1% w / w relative to the amount of wheat flour added. . This solution was then incubated for a period of 16 hours in a shaking incubator at 50 ° C and 100 rpm. The enzyme-treated medium was then cooled to room temperature, transferred to centrifuge containers, and centrifuged at 6,000 rpm for a period of 10 minutes. The supernatant was passed through a 0.2 pm filter paper using a Buchner funnel, and the filtrate was collected for use in the fermentation process. The filtrate was then autoclaved again. The starch hydrolysis procedure was carried out referring to Panagiotopoulos et al. (2009), Gadonna-Widehem et al. (2012) and the enzyme data sheets provided by Sigma.

1.4 Calculation of biomass

The biomass levels of F. venenatum and S. cerevisiae were calculated for each of the samples taken during the fermentations.

The calculation of the weight of the F. venenatum cells was achieved using microfiber filters. A glass microfiber filter was numbered and the mass of the dry filter was recorded. The filter was placed in a Buchner funnel and a 1 ml aliquot of the fermentation sample was passed through the filter. This process was repeated in triplicate for each sample. The filters were then placed in a Petri dish and dried in an oven at 50 ° C for a period of 24 hours. The filters were then reweighed and, if necessary, dried again until a stable reading was observed. Biomass was calculated by subtracting the previously recorded weight of the empty filter. Since the process was carried out in triplicate, the indicated value is the average of the three recorded weights.

Dry cell weight calculation of S. cerevisiae was performed using Eppendorf tubes. The Eppendorf tubes were dried in a desiccator and the weight of the dry and empty tube was recorded. A 1 ml aliquot of the fermentation sample was pipetted into the tube and then centrifuged at 6000 rpm for a period of 10 minutes. The supernatant was removed, and the cell pellet was dried. The tube was reweighed until a stable reading was observed, and the mass of the empty tube was subtracted to produce the mass of cells present. Since a 1 ml aliquot of the fermentation sample was used, the value determined in this case is indicated as the dry cell weight in g / ml. All samples were retested in triplicate, the indicated value being the average of the three replicates.

1.5 Quantification of glucose

The glucose concentration present in each sample was measured using the YSI Biochemistry Analyzer. Early samples required a dilution factor in the region of x10 to be applied, to reduce glucose concentrations to levels that the chemistry analyzer can reproducibly quantify. During Saccharomyces fermentation, late samples can be analyzed directly without the need for dilution steps.

1.6 Quantification of ethanol

The quantification of ethanol in the samples was achieved using a high performance liquid chromatography (HPLC) method. Separation was achieved using a REZEX ROA-H + organic acid chromatography column and a 0.005 N sulfuric acid mobile phase at a flow rate of 1 ml / min. Detection was done by a refractive index detector.

1.7 Culture in shake flasks

Shake flask cultures were performed in 500 ml or 100 ml conical flasks containing 200 ml or 40 ml of medium (MYPG or wheat hydrolyzate with Vogel salts). The flasks were incubated on a vertical shaker set at 150 rpm and 28 ° C for the F. venenatum cultures, and at 30 ° C and 250 rpm for the S. cerevisiae cultures.

1.8 Batch culture in bioreactor

1.8.1 Batch culture of F. venenatum

The batch fermentations of mycoproteins were carried out in two different fermentation systems described below. The fermentation temperature was 28 ° C, and the pH was controlled to 6 with 2M sodium hydroxide. The DO probe was calibrated with nitrogen without oxygen for the O ² value of 0%, and the calibration of the slope with compressed air. The probe calibration was carried out under the fermentation conditions. The inoculum, a 72 hour cultured shake flask culture grown at 28 ° C and 150 rpm on a vertical shaker, constituted 10% v / v of the final fermentation volume. The medium used for the fermentation was N medium (Vogel, 1956) or wheat hydrolyzate supplemented with Vogel salts. In the case of using wheat hydrolyzate as the fermentation medium, rapeseed oil was used as antifoam reagent when necessary.

1.8.1.1 Applikon

The borosilicate vessel of this fermentation system has a total volume of 2L, and the fermentations were performed at a 1.5L working volume with a 2: 1 height-to-diameter ratio. The fermenter is equipped with four moving screens and two six-blade Rushton impellers, powered by an Applikon P100 overhead motor. Aeration was accomplished by bubbling filtered air through a ring bubbler positioned under the impeller. The pH was measured with a Mettler Toledo pH probe and a Mettler Toledo DO probe was used for the determination of dissolved oxygen. An Applikon heating jacket was used to heat the fermenter. The system was also equipped with a temperature probe and a condenser to prevent evaporation. An Applikon Bio-Console ADI 1035 unit was used in combination with the Applikon Bio-Control ADI 1031 control unit to control the parameters. The agitation used with this fermentation system was 600 rpm.

1.8.2 Ethanol fermentation

The ethanol fermentation was carried out in two different fermentation systems described below in detail. The agitation used was 500 rpm, and the temperature was controlled at 30 ° C. The calibration of the pO ² probe was carried out under fermentation conditions. The ^{0% O 2} value was adjusted by aerating with oxygen-free nitrogen, and the probe slope was calibrated with compressed air. The pH was controlled with 2M sulfuric acid and ammonia 25% v / v to a value of 5.5. The fermentation broth was only aerated with 0.82 vvm between 8 and 18 hours of the fermentation time. The inoculum, a shake flask culture grown for 24 hours (30 ° C, 250 rpm), constituted 5% v / v of the final fermentation volume. To control foaming during fermentation, PPG was added as necessary.

1.8.2.1 Biostat C (C15-3) with DCU 3 (B. Braun Biotech)

The stainless steel fermenter has a total volume of 22 l and a double wall that allows heating or cooling of the fermenter. It has a side viewing window and is equipped with four screens, as well as three six-blade Rushton impellers that are above a ring bubbler. The pO ² probe, as well as the pH probe, are from Mettler Toledo. The ratio of height to diameter is 3: 1, with a diameter of 21 cm and a height of 57 cm. The DCU-3 unit allows you to control and monitor the fermentation. The pH was controlled with the help of acid and base pumps. In addition, the system is equipped with a defoamer pump that was used when necessary.

1.8.2.2 DASGIP (Eppendorf)

The fermentation system allows the realization of four parallel fermentations, which can be controlled independently. The flat-bottomed fermenter vessels have a diameter of 9 cm and a height of 24 cm, which is equal to a 3: 1 height-to-diameter ratio. The containers have a working volume of 400-1500 ml. The suspended motor churns the two six-bladed Rushton impellers. Aeration is achieved by bubbling air through a sterile filter through an L bubbler. The system is fully equipped with a main unit that heats the vessels, acid and base pumps, pH and pO ² detection units, detector of temperature, emission gas analysis, and agitation control.

Results

1.9 Wheat medium

The wheat medium used for this fermentation was obtained following the indicated procedure (1.3). This particular medium preparation uses the batch of wheat identified as 'RCS' with a load of 146 g of wheat flour dissolved in 2 L of distilled water. In the process, an enzyme load of 1.46 g of α-amylase and 1.5 g glucoamylase.

The glucose concentrations mediated using the YSI biochemical analyzer at the various stages in the preparation of the medium are listed in Table 1.

Table 1 - Glucose concentrations at various stages during the enzymatic treatment of wheat medium

The following experiments were performed to support the invention.

Table 2 - Experiments supporting the invention

1.10 Experiment 1 (comparative example)

1.10.1 Aims and objectives

The objective of this experiment was to investigate the viability of cultivating F. venenatum for the production of mycoproteins using a carbohydrate raw material (wheat hydrolyzate).

1.10.2 Experimental conditions

Shake flask cultures of F. venenatum were grown initially in wheat hydrolyzate and in glucose (both media supplemented with Vogel salts) to compare the growth profile in the two different media. The flasks were assembled and analyzed in triplicate. Samples were taken after 72 hours and analyzed for biomass and glucose content (9).

The fermentation configuration for the mycoprotein fermentation was: aeration with 1 slpm and adjustment of the stirrer speed to 1000 rpm. The temperature was maintained at 28 ° C, the pH was adjusted to 6, and the fermenter was inoculated with a 72 h shake flask culture of 10% v / v of the final fermentation volume. Wheat hydrolyzate for fermentation was supplemented with Vogel salts. The first fermentations revealed the problem of strong foaming at the beginning of the fermentation. This led to the accumulation of biomass in the foam and, in conclusion, growth only occurred in the upper part of the fermentation broth, but not in the solution. Foaming was probably caused by proteins in the wheat hydrolyzate, and since the minimal Vogel's medium used in the previous fermentations did not contain protein, foaming was not a problem in the previous batch fermentations and, furthermore, it is not a problem in industrial processes used today.

To solve this problem, rapeseed oil is used as an antifoam reagent. Rapeseed oil was chosen because it can be used in a food grade product and would be an inexpensive solution. It can also be used as a carbon source by Fusarium, which would lead to higher yields, and since foaming is only a significant problem early in the process, this will not be a problem at later times.

Since the use of rapeseed oil as an antifoam reagent alone could not solve the problem of inoculum accumulation at the top of the liquid level in the fermenter, a stirring and aeration profile was used. In detail, this means that agitation and aeration slowly increased over a time of 1.5 hours. The agitation and aeration rates used are shown in the following table.

Table 3 - Profile of agitation and aeration of the discontinuous fermentation of F. venenatum with wheat hydrolyzate The moments that the parameters increase are shown.

1.10.3 Results

With the use of this profile, a discontinuous fermentation was carried out with success, which can be observed in 10. The maximum biomass reached in this fermentation was 8.97 g / l, which, taking into account a starting glucose concentration of 49 , 46 g / l, produces a yield of 18.1%. This achieved yield is only half of the yield that was achieved in batch fermentation with minimal Vogel's medium. This is probably due to a strong oxygen limitation at the beginning of fermentation that crucially restricts growth. Dissolved oxygen decreases rapidly at the start of fermentation, since aeration and agitation are very low at the beginning, which is disadvantageous for oxygen transfer to the fermentation broth (data not shown). A higher mycoprotein yield could probably be achieved by improving oxygen supply, since 9.98 g / l ethanol was also produced as a by-product during fermentation. The production of these high levels of ethanol was not expected during this fermentation, and further experiments will be conducted to investigate the effect of culture conditions on the co-production of ethanol and mycoproteins by F. venenatum (section 1.13).

1.10.3.1 Growth of F. venenatum in partially hydrolyzed medium (no enzymes are used)

Due to certain considerations about the economy of the process and the high costs in the acquisition of hydrolyzing enzymes and the high costs of the hydrolysis process due to the heating / cooling stages and the pH adaptations, the use of flour was taken into account. wheat, which was only dissolved in water, or partially hydrolyzed wheat, performing only the liquefaction stage. To investigate the influence of this on the growth of the Fusarium strain, it was cultivated in shake flasks in different hydrolysates supplemented with Vogel salts. For this purpose, 70 g / l of wheat flour were dissolved in water at 90 ° C and then only the liquefaction stage was carried out with 0.5% w / w of α-amylase in one sample, and in the other sample, both liquefaction and saccharification steps were performed with the additional use of 1% w / w glucoamylase. A third did not contain enzymes. All three media were analyzed by HPLC and used for shake flask experiments.

HPLC analysis of the different hydrolysates showed that only the hydrolyzate with both liquefaction and saccharification stages contained glucose, and 70 g / l of wheat flour produced 48 g / l of glucose. The experiment was performed in triplicate in 500 ml shake flasks containing 200 ml of medium supplemented with Vogel salts, and samples were taken after 24, 48 and 72 hours and analyzed in triplicate.

The results of this experiment demonstrate that F. venenatum can grow equally well in a medium consisting only of wheat hydrolyzate with added Vogel salts (11). Therefore, it is assumed that it uses starch in solution as a carbon source. Growth is slightly faster in the fully hydrolyzed medium, which is probably caused by the need for amylase production in Fusarium to use starch as an energy source. The HPLC data (not shown) suggest that even in the enzyme-free medium, disaccharides are present that can be used at the beginning of the incubation and that may be the reason that no differences in growth are observed in the first 24 hours. . After 72 hours, the biomass in all flasks was approximately 7 g / L dry biomass, without noticeable differences.

1.11 Experiment 2 (comparative example)

1.11.1 Aims and objectives

To demonstrate that the integration of mycoprotein fermentation processes and ethanol fermentation is possible, preliminary experiments were performed to demonstrate that S. cerevisiae grows on a wheat hydrolyzate (section 1.10.3.1). Yeast was also cultured in a mycoprotein fermentation filtrate to observe for growth and to determine if F. venenatum produces any substances that inhibit the growth of S. cerevisiae (section 1.11.3.2).

1.11.2 Experimental conditions

1.11.3 Results

1.11.3.1 Growth of S. cerevisiae in a wheat hydrolyzate

Shake flask experiments were performed. For this purpose, 100 ml shake flasks were filled with 40 ml of medium and cultured for 24 h at 250 rpm and 30 ° C. The flasks were cultured and analyzed in triplicate. The results of the OD and the determination of the dry biomass are shown in 2.

The results of this experiment demonstrate that S. cerevisiae grows equally well in hydrolyzed wheat that had been supplemented with Vogel salts and, therefore, integration of the processes is possible. It can be assumed that the wheat hydrolyzate contains proteins and other nutrients that are essential for the growth of S. cerevisiae, since the medium containing only glucose and Vogel salts does not stimulate the same growth.

1.11.3.2 Batch fermentation of S. cerevisiae with a wheat hydrolyzate

To demonstrate that the integration of the two fermentation processes is possible, a fermentation of S. cerevisiae was carried out with a wheat hydrolyzate. The fermentation system used was DASGIP (Eppendorf) which allows the execution of four fermentations at the same time. Two fermentations were carried out with MYPG as a control and two fermentations with wheat hydrolyzate (flour 35 g / l, α-amylase at 0.5% w / w, glucoamylase at 1% w / w) supplemented with Vogel salts. The working volume was 800 ml, and the fermentation was carried out for 24 hours, while the aeration at 0.82 vvm was only present between the 8 to 18 hours of the fermentation time. Yeast growth was monitored by measuring the optical density and determining the dry biomass.

Both the optical density trend and the dry biomass trend in the four fermentations did not show notable differences throughout the fermentation development, 2 and 3.

HPLC analysis of the samples produced the following values for the initial glucose concentrations and the final ethanol concentrations. In addition, the efficiency of the conversion of glucose to ethanol was calculated. The yields of the fermentations with wheat hydrolyzate were slightly higher, but, in general, the trend of both fermentations was similar.

Table 4 - Analysis of glucose concentration at the beginning of S. cerevisiae fermentation and maximum ethanol concentration. The yield of the conversion of glucose to ethanol was calculated.

1.12 Experiment 3

1.12.1 Aims and objectives

The objective of this experiment was to demonstrate the viability of an integrated bioprocess for the production of mycoproteins ( F. venenatum) and ethanol (using S. cerevisiae) using expired food grade wheat as growth medium. Previous experiments carried out suggested that Fusarium and Saccharomyces can be successfully grown in hydrolyzed wheat medium (section 1.10 and 1.11). This experiment builds on these findings, and attempts to grow Fusarium to a point where sufficient biomass is obtained, by the same time that glucose is still present in the system to be used later by S. cerevisiae.

1.12.2 Experimental conditions

Fermentation process

The experimental conditions are detailed in table 5.

Table 5 - Experimental conditions for experiment 3.

1.12.3 Sampling and results

Samples were withdrawn from the culture medium immediately after inoculation and then at various times throughout the fermentation.

The glucose concentrations for each of the collected samples were calculated using the YSI biochemistry analyzer (1.5), as well as the biomass levels of each organism (1.4). Since this analysis was performed in triplicate, the indicated values are the average of the three replicates (5 and Figure 16).

The quantification of ethanol in the samples was achieved using the HPLC method (1.6), and the results are also detailed in 6.

1.12.4 Analysis

This experiment demonstrates the ability to integrate the two fermentation processes of interest. The first stage demonstrates the ability to grow F. venenatum up to a biomass level in the region of 10 g / l. This biomass is then harvested, and the medium, which still contains glucose in the 20 g / l region, is resterilized. After this medium is inoculated with S. cerevisiae and later the growth of this organism is achieved. After a period of approximately 16 hours, the culture was deprived of oxygen to stimulate ethanol production. Fermentation was continued until reaching a total fermentation time of approximately forty hours, and samples were taken at various times for ethanol quantification using the HPLC method developed (1.6).

No ethanol was detected during the fermentation of Fusarium venenatum (data not shown). After inoculation and initial growth of S. cerevisiae, ethanol was detected in the samples (approximately 5 g / l), 6. This demonstrates the viability of the strategy to integrate the two bioprocesses: the ermentation of Fusarium venenatum for mycoprotein production, followed by S. cerevisiae fermentation and ethanol production using expired food grade wheat as growth medium. Process optimization to produce maximum mycoprotein and ethanol yields from fermentations should be performed to improve the overall efficiency of the process.

1.12.5 Conclusions

The results have shown that the integration of the two bioprocesses for the production of the desired products is possible.

1.13 Experiment 4

A series of fermentations were performed to try to demonstrate the viability of a bioprocess for the production of mycoproteins (as food) and ethanol (as fuel) with Fusarium venenatum using expired food grade wheat as the growth medium.

1.13.1 Aims and objectives

The objective of this experiment was to demonstrate the viability of a bioprocess using Fusarium venenatum for the production of mycoproteins and ethanol using hydrolyzed food grade wheat as a substrate.

Previous experiments suggested that Fusarium venenatum produces biomass or ethanol depending on the aeration profile used. For this purpose, three different aeration profiles were used to determine the differences in biomass and ethanol production. A control fermentation with defined Vogel medium (VM) was used and aerated throughout the entire fermentation.

In the first 20 hours of fermentation time, the four fermentations were aerated with the same cascade without oxygen limitation. After 20 hours, fermentations one (VM1) and two (WH2) continued to aerate, while in fermentation three (WH3) oxygen was limited by adjusting the aeration to 0.1 vvm after 20 hours of fermentation. In fermentation four (WH4) the aeration was turned off, which led to oxygen deprivation. The applied conditions are shown in table 6.

Table 6 - Summary of each fermentation condition used to investigate the influence of oxygen limitation

1.13.2 Experimental conditions

The wheat hydrolyzate that was used in fermentations two, three and four was prepared according to 1.3.

1.13.3 Sampling and results

Samples of the culture medium were taken before inoculation and immediately after inoculation. The samples were taken twice daily throughout the fermentation. The details of the samples, the time of taking them and the various controlled process parameters were recorded.

The glucose concentrations for each of the collected samples were calculated using the YSI biochemistry analyzer (1.5), as well as the biomass levels of each organism (1.4). Since this analysis was performed in triplicate, the values indicated are the average of the three replicates. The quantification of ethanol in the samples was achieved using the HPLC method described in 1.6.

1.13.3.1 Fermentation 1

Fermentation one was performed without oxygen limitation for Fusarium venenatum. This fermentation was used as a control, and therefore Vogel's medium with glucose as a carbon source was used as the fermentation medium, instead of wheat hydrolyzate. The objective of this was to compare the growth between the different media and determine the possible differences. It is expected to get more biomass in the fermentations without oxygen limitations and not to detect considerable amounts of ethanol.

1.13.3.1.1 Experimental conditions

The experimental parameters used for this fermentation process are indicated in table 7. This fermentation acts as a control and is compared with fermentation two.

Table 7 - Experimental conditions for fermentation one

1.13.3.1.2 Results

In fermentation one, most of the biomass was produced in the first 20 hours of fermentation, when it reached levels of 22 g / l of dry biomass. The maximum of dry biomass was reached at the end of the fermentation after 96 hours, when 25 g / l of dry biomass were present. Ethanol was not detected throughout the fermentation. After 20 hours, only 0.4 g / l of glucose was available from the initial 43.5 g / l for Fusarium. The trends of the levels of dry biomass, ethanol and glucose can be observed in 7, 8 and 9. 1.13.3.1.3 Conclusions

As expected, no ethanol was produced in this fermentation, whereas a large amount of biomass of 25 g / l was obtained. Biomass production can probably be increased by introducing substrate into the fermenter and harvesting the biomass. As the biomass increases, a higher viscosity can be observed that prevents mixing and oxygen transfer. Therefore, the collection of biomass can be beneficial for biomass production.

1.13.3.2 Fermentation 2

The substrate used in fermentation two was hydrolyzed wheat supplemented with Vogel salts, which was also used in fermentation three (1.13.3.3) and four (1.13.3.4). Throughout the fermentation, oxygen was not limited. This fermentation was compared to fermentation one to draw conclusions about the differences in performance between the media used. More similar results were expected from this fermentation, compared to one fermentation with Vogel's medium.

1.13.3.2.1 Fermentation process

In fermentation two, wheat hydrolyzate was used as the fermentation medium. The set point for dissolved oxygen was adjusted to 30%. The other experimental conditions are detailed in table 8.

Table 8 - Experimental conditions for fermentation two

1.13.3.2.2 Results

The trends of dry biomass, ethanol and glucose of the fermentation with wheat hydrolyzate and without oxygen limitation are shown in 7, 8 and 9. The maximum concentration of dry biomass of 21 g / l was reached after 44 hours , although glucose levels reached 0.13 g / l after 27 hours. Ethanol was only present after 20 hours, but it was probably used as a carbon source afterwards and no ethanol could be detected in the further development of the fermentation.

1.13.3.2.3 Conclusions

The fermentation produced approximately the same biomass levels as the first fermentation. The initial glucose levels were lower, which explains the difference in the biomass levels reached. Furthermore, no ethanol was detected throughout the fermentation, with the exception of the 20 hour sample.

1.13.3.3 Fermentation 3

In fermentation three, the effect of oxygen limitation on ethanol production was investigated. The results were compared with previous fermentations, in particular with fermentation with oxygen deprivation conditions (fermentation four). This should increase knowledge about the influence of the presence of oxygen on ethanol production.

1.13.3.3.1 Experimental conditions

The experimental conditions are detailed in table 9. A wheat hydrolyzate supplemented with Vogel salts was used as the fermentation medium and, after a phase of 20 hours without oxygen limitation, the aeration was adjusted to 0.1 vvm to obtain oxygen limiting conditions.

Table 9 - Experimental conditions for fermentation three

1.13.3.3.2 Results

In fermentation three, the initial glucose concentration was 34.5 g / L, and glucose was stripped from the fermentation medium after 27 hours. The biomass produced was much lower than in the fermentations without oxygen limitation (fermentation 1 and 2), detecting a maximum value of 11 g / l after 75 hours of fermentation time. The ethanol production reached its maximum value of 4.8 g / l after 44 hours. Trends for substrate and products throughout fermentation can be seen in 7, 8 and 9.

1.13.3.3.3 Conclusions

In fermentation three (under oxygen limiting conditions) the highest amount of ethanol was produced during the first 24 h under oxygen limiting conditions. During the later stages of fermentation, the concentration of ethanol decreased, suggesting that it is being used as a carbon source by the body.

1.13.3.4 Fermentation 4

In fermentation four, the amount of oxygen deprivation to the organism was studied after an initial growth phase of 20 hours that would affect the amount of ethanol produced. Lower biomass levels are expected to be achieved during the oxygen limiting phase, but a considerable increase in ethanol production.

1.13.3.4.1 Experimental conditions

The experimental conditions used for fermentation four are detailed in Table 10. A wheat hydrolyzate was used as the fermentation medium and, after 20 hours without oxygen limitation, the aeration was adjusted to 0 vvm to deprive Fusarium of oxygen.

Table 10 - Experimental conditions for fermentation four

1.13.3.4.2 Results

Samples were taken at regular intervals throughout the fermentation process. Fermentation four presented an initial glucose concentration of 32.5 g / l and this was cleared after 27 hours. The dry biomass also reached a maximum after 27 hours (10 g / l). Ethanol concentrations were highest at the end of fermentation and reached 11.7 g / l. The detailed change of the determined parameters is shown in 7, 8 and 9. 1.13.3.4.3 Conclusions

The results of this fermentation suggest that oxygen deprivation is advantageous for ethanol production. After switching to stop aeration, the biomass did not increase further, but decreased until the end of the fermentation. Although the biomass concentration decreased, ethanol continued to increase during the deprivation phase of the fermentations and reached its maximum levels at the end of the process. Glucose was cleared after 27 hours, and it is possible that other oligosaccharides present in the wheat hydrolyzate were used for ethanol production.

1.13.3.5 Conclusions of experiment 4

To understand the effect of the different aeration profiles on biomass and ethanol production, the parameters of the fermentation process were compared. In 7 it can be seen that it is very evident that fermentations with limitation and deprivation of oxygen produce significantly less dry biomass than fermentations without oxygen limitation. However, during the aerated phase of wheat hydrolyzate fermentations (first 20 h), the growth rate is approximately the same (0.34 g / l.h), which demonstrates the reproducibility of the process.

In addition, the production of the second product, ethanol, in the different fermentations was investigated. In these fermentations it was observed that oxygen deprivation leads to a significantly higher ethanol production (8), which is significantly different from the ethanol levels achieved in the other fermentations. After 20 hours, all fermentations carried out with wheat hydrolyzate (fermentations 2-4) showed the same amount of ethanol, which is different from the fermentation carried out with Vogel's medium (fermentation 1). Only fermentation with oxygen deprivation showed ethanol in the fermentation medium at the end of the fermentation. In 8 the glucose concentrations are shown against the development of fermentation. The starting glucose concentrations in the wheat hydrolyzate were slightly lower than in the Vogel's medium, although the consumption rate in all fermentations was approximately 0.40 g / l * h. Biomass and ethanol yields were calculated during the aerated phase (table 11) and during the entire process time (table 12) to obtain a better comparison of the fermentations.

Table 11 - Process performance during the aerated phase of fermentation (first 20 h) for the conversion of glucose into biomass and ethanol, as well as the productivity of biomass and ethanol

Abbreviations: VM1: Vogel's medium, without oxygen limitation; WH2: wheat hydrolyzate, without oxygen limitation, WH3: wheat hydrolyzate, with oxygen limitation; WH4: Wheat hydrolyzate, oxygen deprived. And ^{dcw / gic} : yield of biomass to glucose, YEtOH / Glc: yield of ethanol to glucose, PDCW: productivity of biomass, PEtOH: productivity of ethanol

As expected, during the aerated phase, fermentation one (VM1) showed a higher conversion of biomass to glucose than fermentations using wheat hydrolyzate (fermentations two-four). Biomass productivity was also higher by a factor of 3 in Vogel's medium (fermentation one, VM1), indicating that glucose is more efficiently converted to biomass in defined medium (VM). However, ethanol production is favored in the wheat hydrolyzate medium (fermentations two-four), even during this phase of the process.

In table 12 it can be seen that, although the biomass levels during fermentation two were lower than in fermentation one, the overall yield of the conversion of glucose into biomass is similar (approximately 50%). Furthermore, the highest conversion of glucose to ethanol and the highest productivity of ethanol occur under conditions of oxygen deprivation (WH4). This means that these conditions favor ethanol production compared to biomass production (higher under conditions without oxygen limitation).

Table 12 - Total process yields for the conversion of glucose into biomass and ethanol, as well as the productivity of biomass and ethanol (overall process time of 96 h).

Abbreviations: VM1: Vogel's medium, without oxygen limitation; WH2: wheat hydrolyzate, without oxygen limitation, WH3: wheat hydrolyzate, with oxygen limitation; WH4: Wheat hydrolyzate, oxygen deprived. Y ^{dcw / gic} : biomass yield to glucose, YEtOH / Gic: ethanol yield to glucose, P ^dcw : biomass productivity, PEtOH: ethanol productivity

In addition to controlling the substrate and product concentrations, other process parameters were also controlled. Dissolved oxygen concentration is displayed providing information about oxygen levels in the fermentation medium. Fermentations three and four were oxygen limited after 20 hours, when aeration was decreased.

1.13.3.6 Conclusions

The results shown in this report demonstrate that the hypothesis of producing biomass, as well as ethanol, with Fusarium venenatum from a hydrolyzed food grade wheat is feasible. In addition, they also demonstrate for the first time that the control of the process (specifically, the manipulation of the aeration conditions) favors the production of biomass (without conditions of oxygen limitation) or the production of ethanol (conditions of oxygen deprivation). The optimization of the process can be carried out in such a way that aeration is present for a sufficient time to obtain a sufficient concentration of biomass, before the organism can deprive itself of oxygen and, therefore, the fermentation of ethanol can begin.

A process with oxygen starvation conditions seems preferable for a high ethanol yield. Even at very low biomass levels a substantial amount of ethanol can be produced (and is favored) in the absence of oxygen (fermentation four). It is possible to collect large amounts of biomass before the start of the ethanol production phase (first 20 h of the process). Therefore, by manipulating the process conditions it is possible to adjust the process to obtain the desired product.

The use of a single organism for the production of both products (biomass and ethanol) has great economic and downstream processing advantages. A production process can be adapted according to the demand for mycoproteins (biomass) or ethanol by applying a prolonged or shortened aeration phase. The process can be further improved by harvesting the biomass and introducing more wheat hydrolyzate into the fermentation. Producing ethanol without the need for aeration can reduce production costs substantially and thus can influence the overall economics of the process.

Bibliographic references

Cardona CA, Sánchez Ó.J., Fuel ethanol production: Process design trends and integration opportunities, Bioresour. Technol., 2007, 98: 2415-2457.

Finn B., Harvey LM, McNeil B., Near-infrared spectroscopic monitoring of biomass, glucose, ethanol and protein content in a high cell density baker's yeast fed-batch bioprocess, Yeast, 2006, 23: 507-517.

Claims (9)

1 An integrated method to produce and isolate mycoproteins produced by Fusarium venenatum and ethanol from a raw material of hydrolyzed starch, said method comprising the steps of: a) providing an aqueous fermentable broth comprising a hydrolyzed starch raw material obtained from one or more cereals; b) fermenting at least a portion of the aqueous fermentable broth with Fusarium venenatum to obtain mycoproteins produced by Fusarium venenatum and a partially fermented broth; c) separating the mycoproteins produced by Fusarium venenatum from the partially fermented broth; d) fermenting at least a portion of the partially fermented broth, optionally with a portion of the non-fermented aqueous fermentable broth, with one or more microorganisms to obtain ethanol and a spent fermentation residue; Y e) isolating the ethanol from the spent fermentation residue. 2. - The method according to claim 1, in which the operating conditions are controlled in each of the stages to preferably favor one or the other product. 3. - The method according to claim 1 or 2, in which the mycoproteins produced by Fusarium venenatum are totally or partially isolated between the 2 fermentation stages. 4. - The method according to claim 1, in which the mycoproteins produced by Fusarium venenatum are totally isolated from the first fermentation stage, and a second fermentation stage is carried out to obtain ethanol using a microorganism other than Fusarium venenatum. 5. - The method according to claim 1, in which Fusarium venenatum is used for both stages of production of mycoproteins and ethanol. 6. - The method according to any preceding claim, in which the mycoproteins produced by Fusarium venenatum and ethanol are produced in a single fermenter where the operating conditions are controlled to produce the desired mixture proportion of mycoproteins produced by Fusarium venenatum and ethanol. 7. - The method according to any preceding claim, in which the initial substrate is a grain material, and the integrated process also produces a co-product with a higher protein content that can be used as a source of feed for cattle. 8. - The method according to claim 7, in which the chosen production mixture maximizes the production of mycoproteins produced by Fusarium venenatum and the co-product with a higher protein content. 9. - The method according to any preceding claim, in which the operating conditions and the rate or proportion of the resulting production mixture of mycoproteins produced by Fusarium venenatum and ethanol can be altered to correspond to changes in energy supply or costs. taking into account the higher energy intensity of fermentation under aerobic conditions necessary to increase the production rate of mycoproteins produced by Fusarium venenatum.